Fusarium avenaceum and its use as biological control agent for rubus species

ABSTRACT

The invention disclosure relates to a biologically pure isolate of Fusarium avenaceum ATCC 200684 to a herbicidal composition containing the isolate as active ingredient, and to a method of combating weeds, particularly the Rubus species, comprising applying an effective amount of the composition, thereto.

BACKGROUND OF THE INVENTION

This invention relates to Fusarium avenaceum, to a method for itsproduction, and to its use as a herbicide for Rubus species.

The discovery and development of potential biological control agents tosuppress competing forest vegetation is receiving increased attention inthe management of conifer regeneration sites. Development ofalternatives to commonly used weed control methods, such as herbicideapplications and manual cutting, has become important in forestmanagement plans due to economical constraints and increasing publicconcern over pesticide use. Biological control strategies which utilizemicrobial organisms or their secondary metabolites to control weeds havebeen widely investigated on agricultural crops. In forest renewal sites,biological control agents need to be sufficiently virulent to suppresscompeting vegetation that is often diverse in growth habit and density,while allowing for vegetation to resume its role in forest ecosystemsonce conifer release has been obtained.

Invasive Rubus species, namely wild red raspberry [Rubus strigosusMichx.=R. idaeus var. strigosus (Michx.) Focke], thimbleberry (R.parviflorus Nutt.), and salmonberry (R. spectabilis Pursh), are amongthe top 20 forest weeds in Canada. These native Rubus species caneffectively outcompete newly planted or naturally regenerated conifersin reforestation sites in Canada and the northern United States, andreduce the growth and survival of black and white spruce. These Rubusspecies are perennial, deciduous shrubs which form monospecific,multi-layered shrub communities with long-lived clonal root systems.

DESCRIPTION OF THE PRIOR ART

Previous biological control research to reduce competing Rubus speciesworldwide has employed various approaches: the inoculative strategy ofintroducing exotic pathogens, the inundative strategy of usingindigenous pathogens, and the biorational strategy of using phytotoxicmicrobial compounds. The inoculative approach successfully utilized tworust fungi, Phragmidium violaceum (Schultz) Winter and Kuehneolauredinis (Lk.) Arth., to control exotic or naturalized Rubus species inAustralia, New Zealand, Chile, and Hawaii (Gardner ¹, Bruzzese & Hasan²) Inundative applications of three indigenous fungal pathogens,Septoria rubi West., Cylindrocarpon destructans (Zinf.) Scholten, andHainesia lythri (Desm.) Hohnel, were effective against R. parvifloruswhen inoculum was formulated or when plant resistance was weakened byprior mechanical or chemical wounding (Wall & Shamoun ³, Shamoun &Callan ⁴). The biorational approach has utilized bialaphos, a phytotoxinproduced by Streptomyces viridochromogens, to successfully reduce heightand resurgence of R. strigosus in Picea mariana plantations in easternQuebec (Jobidon ⁵).

SUMMARY OF THE INVENTION

According to one aspect of the invention, a biologically pure isolate ofFusarium avenaceum, having the identifying characteristics of ATCCDeposit no. 200684, is provided.

The isolate was deposited, with the American Type Culture Collection(ATCC), Rockville, Md., 20852, USA, on Sep. 26, 1996, under Accessionno. 200684, and was converted to a Budapest Treaty Deposit on Mar. 3,1998. The viability of the isolate was tested and found viable by theATCC, on Mar. 13, 1998.

According to another aspect of the invention, a herbicidal compositionis provided, containing as active ingredient, Fusarium avenaceum, havingthe identifying characteristics of ATCC Deposit no. 200684.

According to yet another aspect of the invention, a method for combatingweeds is provided, comprising applying to a weed plant, an effectiveamount of a herbicidal composition containing as active ingredient, abiologically pure isolate of Fusarium avenaceum, having the identifyingcharacteristics of ATCC Deposit no. 200684.

Preferably, the active ingredient is provided as an aqueous suspensionof an agriculturally acceptable sterile granular growth substrate,capable of supporting the growth of the fungus, infested therewith, andan adjuvant.

Most preferably, the adjuvant is an organosilicone surfactant, soldunder the trademark Silwet L-77, and the substrate is a cereal grain,e.g., rice.

It will be appreciated by those skilled in the art that variousamendments which aid in fungal survival and growth, and facilitatefungal dispersal and adhesion to plant surfaces may also be included,such as nutrients, humectants, invert emulsions and dispersing agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the effect of temperature on Fusariumavenaceum growth, and spore germination.

FIG. 2 is a fulltone illustrating disease symptoms on Rubus strigosusplants after inoculation with Fusarium avenaceum composition accordingto the invention.

FIG. 3 is a fulltone illustrating a comparison of Rubus strigosus plantsinoculated with Fusarium avenaceum composition according to theinvention, versus a control.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods

Isolation and Selection of Fungi

Samples of foliage and stems of R. strigosus, R. parviflorus, and R.spectabilis with disease symptoms such as anthracnose, foliar and stemlesions, necrosis, shoot blight, and dieback were collected from central(49° to 54° latitude) and coastal (including coastal mainland andVancouver Island) British Columbia from May to September, 1990-1994.Samples were obtained from the following biogeoclimatic zones: CoastalDouglas-fir, Coastal Western Hemlock, Interior Cedar-Hemlock, InteriorDouglas-fir, Montane, and Sub-Boreal Spruce. Diseased plant tissues wereexcised (ca. 0.25 cm² sections) and surface-disinfested by successive 1min rinses in 95% ethanol, 0.525% sodium hypochlorite (w/v) and 3 rinsesin sterile distilled water. Tissues were blotted on sterile filterpaper, aseptically plated onto malt extract or potato dextrose agar(MEA, PDA, Difco Laboratories, Detroit, Mich.), and incubated at 20-25°C. with a ca. 12 h light/dark cycle. Resulting fungal colonies weresubcultured from hyphal tips and pure cultures were stored on MEA andPDA slants and in sterile distilled water at 5° C., with periodictesting for viability. For subsequent testing, minimum subculturing wasdone and fungi were inoculated onto Rubus hosts and re-isolated whennecessary.

Twenty isolates were evaluated for pathogenicity by inoculating detachedleaves of Rubus species obtained from shadehouse-grown plants. Testplants were grown from field rootstocks (after cold stratification for 3months at 0° C.) by planting 10 cm-long root segments in a peat-perlite(1:2, v/v) medium and placing in a mist chamber. Healthy plants weretransplanted and maintained at an average height of 0.5 m in 1 gallonpots in peat-vermiculite-sand (3:1:1) medium with a low rate of slowrelease fertilizer (18-7-12 Osmocote, Grace Sierra, Milipitas, Calif.)in an outdoor shadehouse. Additional plants were later propagated fromRubus stem cuttings by dipping 10-cm long stem segments with two leavesin 0.4% indole-3 butyric acid rooting powder (Stim-root No. 2, PlantProducts Ltd., Bramalea, ON) and planting in soil mixtures as above.Mature plants were maintained in the greenhouse at 18-21° C., with ca.60% relative humidity and a 16 h photoperiod.

Detached Rubus leaves were placed on moistened filter paper (9-cmdiameter) in glass Petri plates and inoculated with mycelial plugs (1cm²), taken from 7-day old colonies, with 3 replicates per isolate, andincubated on the lab bench for 7 days. Control leaves were inoculatedwith sterile MEA or PDA plugs under identical conditions and oftenremained green for up to 7 days. Percent leaf area damaged was assessedvisually by using the area-addition method in which percent necrosiswithin leaf quadrants was added cumulatively and the mean percentage wascalculated per leaf. Values>50% were considered to indicate strongpathogenicity. An isolate of F. avenaceum was selected from thesescreening tests after causing >50% leaf area necrosis within 7 days.

Colony Growth and Spore Germination

Temperatures ranging from 0-35° C., in 5° C. increments, were used todetermine optimum colony growth and spore germination. Fungal colonieswere initiated from 5-mm diameter mycelial plugs on PDA and grown underdark conditions for 7 days. For germination tests, conidia were obtainedfrom sporulating colonies on MEA or PDA by flooding plates with steriledistilled water and gently scraping the surface. Conidial suspensionswere diluted and spread onto 2% water agar plates, incubated under darkconditions, and percent germination was recorded at 24 h with a total of300 spores counted at each temperature. For both tests, there were 3-5replicate plates of each fungus at each temperature and the experimentswere repeated.

Inoculum Production

Several agar and liquid media were evaluated for their ability topromote sporulation, as determined by hemacytometer counts, and thefollowing media were selected. Fusarium avenaceum was grown in modifiedRichard's V-8 broth, infested with two mycelial plugs (5 mm) per 250 mLbroth, and maintained on a continuous shaker at 100 rpm at 20-22° C.with a 12 h light/dark regime. Agar plates were infested with onemycelial plug (5 mm) taken from actively growing colonies and incubatedat 20-22° C., with an alternating 12 h light/dark regime.

Grain substrates, namely rice, millet, and barley, were also evaluatedas growth media and were prepared following procedures outlined by Abbaset al. ⁶. Specifically, sterile grains are infested with FA andincubated at 20-22 ° C. under optimum light and humidity conditions,with daily shaking. After 2-5 weeks, grains were air-dried and stored atcool temperature. The grains were then ground to a coarse powder (0.1 to5 mm), resuspended in sterile water (optimum 5 g of substrate per 50 mlof water), sonicated and filtered to obtain culture filtrates forsubsequent use.

Pathogenicity Tests

Inundative applications of conidial inoculum were made to Rubus plantsexposed to exterior temperature and light conditions in shadehousetrials. Inoculum of each fungus consisted of 10⁶ spores/mL combined with2% sucrose and 0.5% gelatin and was sprayed onto test plants with ahand-held sprayer (Garden Sprayer, Greenleaf Products Inc., Burnaby, BC)at a rate of 50 mL/m². A 24 h dew period, provided by covering plantswith a clear plastic bag, was included to enhance germination andinfection. Plants were rated for extent of necrosis by dividing theplant into quadrants, adding cumulatively the percent necrotic area perquadrant, and calculating the mean percent necrosis per plant. Foliarnecrosis was determined on a scale of 0-4 where: 0=no injury, 1=<1%injury, 2=1-10% injury, 3=11-50% injury, 4=51-100% injury. An injuryindex was subsequently calculated as follows: (summation of [severityratings×#plants in that class])/total #plants (Yang et al. ⁷) withratings of <2 indicating slight injury, 2-3.5 indicating moderateinjury, and >3.5 indicating severe injury. Plants were rated for up to 3weeks after inoculation and compared to control treatments of water,with 2-3 replicate plants per treatment, and the experiment wasrepeated. Re-isolations of fungi from treated plants was attemptedfollowing methods described previously. Treatment data were combined andsubjected to one-way analysis of variance to test for differencesbetween treatment means, followed by the Student-Newman-Keuls test atP=0.05.

ADJUVANTS

Adjuvants were included in the inoculum in an effort to increasepathogenicity. Adjuvants were evaluated individually when combined withspore suspensions (10⁶ spores/mL) produced on media as described above.Amended inoculum was applied as a central, 100 μL drop onto detachedRubus leaves, incubated as described for pathogenicity screening tests,and evaluated for necrotic area over 7 days. Inoculum viability wasverified by plating on MEA or PDA. Control treatments consisted of sporesuspensions or adjuvants, and water. Fusarium avenaceum was grown inliquid media and combined separately with 1% malt broth (DifcoLaboratories, Detroit, Mich.), 1% neopeptone (Sigma Chemical Co., St.Louis, Mo.), and 1% sodium alginate (BDH Inc., Toronto, ONT), or grownon rice media and combined with 0.2% v/v and 0.4% v/v Silwet L-77^(R)(organosilicone surfactant, Loveland Ind., Greeley, Colo.). Among all ofthese formulations, those which resulted in >50% necrosis were furthertested on intact plants as described above. Tests on whole plants wererepeated, with 2-3 replicate plants per test, and treatment results weresubjected to statistical analysis as described above.

For use with a granular substrate, culture filtrates from above werecombined with an organosilicone surfactant e.g. Silwet-L-77, or otheradjuvants to produce an inoculum. In this test, FA+0.4% v/v Silwet (seeTable 1, below) was effected. The inoculum was sprayed onto Rubus plantsat 50 ml/m² . Foliar necrosis was determined as described above.

Amendment with Glyphosate

The effect of glyphosate (Roundup®, Monsanto Canada Inc., Sardis, BC) onfungal growth on PDA was determined by adding concentrations of up to 6mM glyphosate or 0.06% filter-sterilized (0.2 μM) Roundup®. Amendedplates were inoculated with a mycelial plug (5 mm) taken from activelygrowing colonies of the three fungi and incubated at 20° C. Colonydiameter was measured at 7 days from 4-6 replicate plates per treatmentand the experiment was repeated. Colonies were further assessed forconidial germination after 2-3 weeks, as previously described.

In shadehouse trials, R. parviflorus and R. spectabilis were treatedwith glyphosate [2 mM or 6 mM glyphosate (0.02% v/v or 0.06% v/vRoundup®)] (10-fold less than the recommended dose of Roundup® for Rubusspecies), applied at 50 mL/m², followed by inoculation after 24 hr withspore suspensions (10⁶ spores/mL, obtained from colonies in liquid andagar media) amended with 0.02% v/v Tween 80, applied at the same rate.Plants were visually rated for percent necrotic leaf area over a 3-weekperiod as described previously and compared to control treatments ofspore suspensions, glyphosate, or water. For each treatment, threereplicate plants were included and the experiment was repeated and datacollected was subjected to statistical analysis as described above.

Results and Discussion

The cultures of F. avenaceum isolated from field collections and testedon detached Rubus leaves, caused >50% necrosis in 7 days. Fusariumavenaceum was collected from stem lesions on R. strigosus in theSub-Boreal Spruce biogeoclimatic zone. Maximum colony growth and sporegermination was observed between 10-30° C. and 15-25° C., respectively,for F. avenaceum (FIG. 1).

On Rubus plants in shadehouse trials, F. avenaceum gave sufficient andreproducible foliar necrosis.

Fusarium avenaceum, when grown on rice grains and combined with 0.4% v/vSilwet L-77®, induced greater foliar necrosis on the Rubus spp. than anyother fungus or treatment tested. Treated foliage developed awater-soaked appearance, followed by the development of extensive foliarnecrosis, within 24-48 h on R. strigosus and R. parviflorus. Thisresulted in large areas of necrotic leaf tissue, leaf curl and death(FIGS. 2 & 3). Rubus strigosus was the most susceptible to the F.avenaceum+Silwet treatment, with 89% of plants demonstrating 51-100%injury within 7 d of treatment (Table 1). On R. parviflorus, F.avenaceum+Silwet caused 11-50% injury and 51-100% injury on 44% ofplants, respectively. Only 6.25% of R. spectabilis test plantsshowed >50% injury with a similar treatment. Analysis of variancefollowed by the Student-Newman-Keuls test indicated significantdifferences between the F. avenaceum+Silwet treatment and all othertreatments for R. strigosus (F=61.39, P=<0.001), R. parviflorus(F=38.43, P=<0.001) and R. spectabilis plants (F=12.39, P=<0.001) (Table1, below). With R. spectabilis, increasing the surfactant dosage to 1%v/v Silwet L-77® did not result in increased foliar necrosis. Fusariumavenaceum was re-isolated from necrotic leaf tissue of inoculated plantsof each Rubus spp. and was not isolated from control plants. All treatedplants flushed new leaves by 3 weeks, and the new foliage and stems werefree of necrotic symptoms.

                  TABLE 1                                                         ______________________________________                                        Foliar necrosis of Rubus plants                                                 resulting from inundative applications of                                     Fusarium avenaceum inoculum, originating from                                 infested rice cultures, and combined with                                     an organosilicone surfactant (0.4% v/v Silwet L-77 ® ).                                       Foliar injury*                                          Treatment Rubus strigosus                                                                          Rubus parviflorus                                                                         Rubus spectabilis                            ______________________________________                                        Control-water                                                                           0.44 ± 0.18 d                                                                         0.20 ± 0.13 c                                                                          0.20 ± 0.13 b                               Surfactant     1.89 ± 0.26 b       2.17 ± 0.31 b    0.67 ±                                          0.21 b                                         F. avenaceum   1.33 ± 0.17 c  1.38 ± 0.38 b    0.75 ± 0.25 b                                         F. avenaceum +   3.89 ± 0.11 a 3.31                                       ± 0.18 a 2.00 ± 0.26 a                   surfactant                                                                  ______________________________________                                         *Foliar injury rating index with <2 = slight injury, 2-3.5 = moderate         injury, and >3.5 = severe injury. A oneway analysis of variance comparing     treatment means was performed. Within a column, means ± standard error     of the mean followed by the same letter are not significantly different       according to the StudentNewman-Keuls test at P = 0.05.                   

A preliminary host range test has demonstrated that several economicallyimportant conifer seedlings did not display disease symptoms wheninoculated with the F. avenaceum-Silwet L-77® formulation.

Glyphosate affected fungal growth since the fungi developed irregularcolony margins, compared to the even mycelial margin observed in thecontrols, when grown on PDA amended with up to 6 mM glyphosate. Colonydiameters on PDA reached 50% of controls at concentrations of >1 mMglyphosate after 7 days. Fusarium avenaceum sporulated on PDA with 0-2mM glyphosate and conidia germinated readily at 25° C. When low doses ofglyphosate were applied to intact plants, followed by fungalinoculation, [a temporary or not significantly greater percent necrosison Rubus species was observed (R. parviflorus; F=16.293, P=<0.001; R.spectabilis; F=12.44, P=<0.001) (Table 2, below)] During the treatmentevaluation period (3 weeks), all plants receiving glyphosate showedincreasing necrosis over time with symptoms of chlorosis (particularlyin young leaves), wilting, and low vigour, indicating susceptibility tovery low doses of the herbicide.

                  TABLE 2                                                         ______________________________________                                        Effect of Fusarium avenaceum spore suspensions                                  (10.sup.6 spores/mL, produced in liquid media),                               applied alone and in a delayed application following low                      doses of glyphosate (Roundup ® ),                                         on foliar necrosis of Rubus species.                                                                               Foliar injury*                         Treatment      Rubus parviflorus                                                                          Rubus spectabilis                                 ______________________________________                                        Control-water  0.29 ± 0.18 d                                                                           0.13 ± 0.13 d                                    0.02% Roundup ®   1.71 ± 0.42 bc           1.83 ± 0.31 ab                                      0.06% Roundup ®   2.67 ± 0.33 ab                                           1.40 ± 0.40 bc                              F. avenaceum        1.38 ± 0.38 c            0.50 ± 0.27 cd                                        F. avenaceum + 0.02% v/v 3.60 ± 0.40 a                                    2.50 ± 0.43 ab                                   Roundup ®                                                                 F. avenaceum + 0.06% v/v 3.83 ± 0.17 a 2.71 ± 0.36 a                    Roundup ®                                                               ______________________________________                                         *Foliar injury rating index with <2 = slight injury, 2-3.5 = moderate         injury, and >3.5 = severe injury. A oneway analysis of variance comparing     treatment means was performed. Within a column, means ± standard error     of the mean followed by the same letter are not significantly different       according to the StudentNewman-Keuls test at P = 0.05.                   

Based on this research, F. avenaceum inoculum produced on rice appearsto have several suitable attributes for further evaluation as acandidate biological control agent for Rubus species. These include asuitable production method for large amounts of inoculum, a formulationrequiring no dew period, a spray method for application, and extensivefoliar damage to R. strigosus and R. parviflorus.

BIBLIOGRAPHY

1. Gardner, D. E. 1983. Leaf rust caused by Kuehneola uredinis on nativeand non-native Rubus species in Hawaii. Plant Dis. 67:962-963.

2. Bruzzese, E., and S. Hasan. 1986. Host specificity of the rustPhragmidium violaceum, a potential biological control agent of Europeanblackberry. Ann. Appl. Biol. 108:585-596.

3. Wall, R. E., and S. F. Shamoun. 1990. Experiments on vegetationcontrol with native pathogenic fungi in the southern interior of BritishColumbia. Can. Forest Serv. and BC Min. of Forests, Forest ResourcesDevelopment Agreement Rep. 134., Victoria, BC. 18 pp.

4. Shamoun, S. F., and B. E. Callan. 1992. Hainesia lythri, a possiblebiocontrol agent for thimbleberry (Rubus parviflorus) in BritishColumbia forests. Phytopathology 80:1080. (Abstr.).

5. Jobidon, R. 1991. Potential use of bialaphos, a microbially producedphytotoxin, to control red raspberry in forest plantations and itseffect on black spruce. Can. J. For. Res. 21:489-497.

6. Abbas, H. K, C. D. Boyette, R. E. Hoagland, and R. F. Vesonder. 1991.Bioherbicidal potential of Fusarium moniliforme and its phytotoxin,fumonisin. Weed Sci. 39:673-677.

7. Yang, S. -M., Johnson, D. R., Dowler, W. M., and Connick, W. J., Jr.1993. Infection of leafy spurge by Altemaria altemata and A.angustiovoidea in the absence of dew. Phytopathol. 83:953-958.

We claim:
 1. A biologically pure isolate of Fusarium avenaceum, havingall of the identifying characteristics of ATCC Deposit no.
 200684. 2. Aherbicidal composition, containing as active ingredient, Fusariumavenaceum, having all of the identifying characteristics of ATCC Depositno.
 200684. 3. A composition according to claim 2, wherein the activeingredient is provided as an aqueous suspension of an agriculturallyacceptable sterile granular growth substrate inoculated therewith, andan adjuvant.
 4. A composition according to claim 3, wherein the adjuvantis an organosilicone surfactant, included in an amount of 0.2 to 0.4%v/v.
 5. A composition according to claim 4, wherein the amount of theadjuvant is about 0.4% v/v.
 6. A composition according to claim 4,wherein the growth substrate is a cereal grain.
 7. A compositionaccording to claim 6, wherein the cereal grain is rice.
 8. A compositionaccording to claim 3, wherein the growth substrate is a cereal grain ofa particle size in the range of 0.1 to 5 mm.
 9. A composition accordingto claim 4, additionally comprising glyphosate in an amount of 0.02 to0.06% v/v.
 10. A method for combating weeds of the Rubus species,comprising applying thereto, an effective amount of a herbicidalcomposition containing as active ingredient, a biologically pure isolateof Fusarium avenaceum, having all of the identifying characteristics ofATCC Deposit no.
 200684. 11. A method according to claim 10, wherein theactive ingredient is provided as an aqueous suspension of anagriculturally acceptable sterile growth substrate inoculated therewith,and an adjuvant, included in an amount of 0.2 to 0.4% v/v.
 12. A methodaccording to claim 11, wherein the adjuvant is an organosiliconesurfactant.
 13. A method according to claim 12, wherein the amount isabout 0.4% v/v.
 14. A method according to claim 10, wherein thecomposition is applied to the weed, post emergence, as a foliar spray.15. A method according to claim 11, wherein the aqueous suspensioncomprises about 5 g of inoculated substrate per 50 ml of water.
 16. Amethod according to claim 15, wherein the aqueous suspension is appliedin an amount of about 50 ml/m², post emergence, as a foliar spray.
 17. Amethod according to claim 11, wherein the weed is Rubus strigosus orRubus parviflorus.
 18. A method according to claim 11, wherein thegrowth substrate is rice.
 19. A method according to claim 11, whereinthe composition additionally comprises glyphosate in an amount of 0.02to 0.06% v/v.